Anti-c-met antibody drug conjugates

ABSTRACT

The present disclosure provides c-Met antibody drug conjugates (ADCs), including compositions and methods of using such ADCs.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 63/181,963, filed Apr. 29, 2021, which is hereby incorporated by reference in its entirety.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 28, 2022, is named 632US_SL_ST25.txt and is 22,918 bytes in size.

3. TECHNICAL FIELD

The present application pertains to, among other things, novel topoisomerase inhibitor drugs, drug linkers, anti-c-Met antibody drug conjugates (ADCs), and methods of making the same.

4. BACKGROUND

c-Met is a signaling tyrosine kinase receptor expressed on the surface of epithelial and endothelial cells. Activation of c-Met by hepatocyte growth factor (HGF), its only known ligand, has been shown to control cell proliferation, angiogenesis, survival, and cellular motility.

Non-small cell lung cancer (NSCLC) represents 85% of all lung cancers and is the leading cause of cancer-related death worldwide. Aberrant c-Met signaling is common in NSCLC and is believed to occur via multiple mechanisms. Deregulated c-Met signaling has been associated with poor prognosis, tumorigenesis, resistance to chemotherapy/radiotherapy, and acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI).

Antibody drug conjugates (ADCs) represent a relatively new class of therapeutics comprising an antibody conjugated to a cytotoxic drug via a chemical linker. The therapeutic concept of ADCs is to combine binding capabilities of an antibody with a drug, where the antibody is used to deliver the drug to a tumor cell by means of binding to a target surface antigen, including target surface antigens that are overexpressed or amplified in the tumor cells.

However, no antibody drug conjugates have been approved for the treatment of non-small cell lung cancer. There remains a need in the art for antibody drug conjugates that can be used for therapeutic purposes, such as in the treatment of non-small cell lung cancer.

5. SUMMARY

The present disclosure provides antibody drug conjugates that specifically bind to human c-Met. The amino acid sequences of exemplary CDRs, as well as the amino acid sequence of the V_(H) and V_(L) regions of the heavy and light chains of the antibody of exemplary anti-c-Met ADCs are provided in the Detailed Description below.

In another aspect, the present disclosure provides compositions including the anti-c-Met ADCs described herein. The compositions generally comprise one or more anti-c-Met ADC as described herein, and one or more excipients, carriers, or diluents.

The present disclosure provides methods of treating subjects, such as human subjects, having NSCLC comprising administering an effective amount of an anti-c-Met ADCs disclosed herein. An anti-c-Met ADC is typically administered as an intravenous infusion and/or injection.

6. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a synthetic pathway for the topoisomerase I (TOP1) inhibitor of Formula I.

FIGS. 2A-2B show cell proliferation following administration of TOP1 inhibitor compounds for Calu-6 (lung adenocarcinoma; FIG. 2A) and A375 (malignant melanoma; FIG. 2B).

FIGS. 3A-3J show in vitro activity of an ADC of the present disclosure (ADC-1) on cell lines, including (A) SNU-5 (human gastric carcinoma), (B) Hs 746T (gastric adenocarcinoma), (C) EBC1 (human lung squamous cell carcinoma), (D) NCI-H441 (human lung adenocarcinoma), (E) NCI-H1573 (lung adenocarcinoma), (F) HCC827 (lung adenocarcinoma), (G) NCI-H820 (lung papillary adenocarcinoma), (H) Calu-3 (lung adenocarcinoma), (I) MIA PaCa-2 (pancreatic ductal adenocarcinoma), and (J) HEK-293.

FIGS. 4A-4B show in vivo activity of ADC-1 in the NCI-H441 (human adenocarcinoma, FIG. 4A), and Hs 746T (gastric adenocarcinoma, FIG. 4B) xenograft tumor models.

7. DETAILED DESCRIPTION

Various aspects of the invention relate to topoisomerase I (TOP1) inhibitors and anti-c-Met antibody drug conjugates comprising such TOP1 inhibitors. In certain embodiments, the invention provides anti-c-Met ADCs, including anti-c-Met ADCs comprising TOP1 inhibitors, synthons useful for synthesizing the ADCs, methods of making the ADCs, and various methods of using the ADCs.

7.1. Topoisomerase 1 Inhibitors (TOP1i)

Topoisomerase 1 (TOP1) removes supercoils formed during DNA replication. TOP1 inhibitors (TOP1i) can bind and stabilize TOP1-DNA complexes, inducing DNA strand breakage and apoptosis. Presented herein is a topoisomerase I inhibitor drug (“TOP1i drug”) according to structural formula (I), which may be purposed for targeted delivery to cells by conjugation to an antibody.

In embodiments, a TOP1i drug is a compound according to formula (I). In embodiments, the TOP1i drug is (7S)-14-(3-aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H, 10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinoline-8,11 (7H,13H)-dione, or a structurally equivalent form thereof.

TOP1i drugs as contemplated herein may be conjugated to an antibody in an ADC as shown in structural formula (II):

wherein

represents the point of attachment of a linker to the TOP1i drug.

7.2. Anti-c-Met ADCs

Topoisomerase inhibitors as described herein may be conjugated to an anti-c-Met antibody to form an anti-c-Met TOP1i antibody drug conjugate (ADC). Antibody-drug conjugates may increase the therapeutic efficacy of antibodies in treating disease due to the ability of the ADC to selectively deliver one or more drug moiety(s) to target tissues, such as a tumor-associated antigen, e.g., c-Met expressing tumors. Thus, in embodiments, the present disclosure provides anti-c-Met TOP1i ADCs for therapeutic use, e.g., in the treatment of non-small cell lung cancer.

In certain embodiments, a TOP1i drug is conjugated to an antibody by way of linker according to structural formula (III):

wherein X represents bromo or an N-linked maleimide.

In the anti-c-Met ADCs described herein, TOP1i drugs are conjugated to the anti-c-Met antibody by way of a linker moiety. As will be appreciated by skilled artisans, the linkers connect the TOP1i drug to the anti-c-Met antibody by forming a covalent linkage to the TOP1i drug at one location and a covalent linkage to the antibody at another. The covalent linkages are formed by reaction between functional groups on the linker and functional groups on the TOP1i drug and the anti-c-Met antibody.

Synthetic intermediate compounds that may be used to form ADCs may include:

7.2.1. Linker Drug LD1 (Structural Formula (V))

(2S)-2-(2-bromoacetamido)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

7.2.2. Linker Drug LD2 (Structural Formula (VI))

(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

7.2.3. Linker Drug LD3 (Structural Formula (VII))

(2S,3S,4R,5R,6S)-6-[2-(5-{[(2S)-2-{(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-3-methylbutanoyl}amino)propanoyl]amino}-2-{[({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyran[3′,4′: 6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}carbamoyl)oxy]methyl}phenyl)ethyl]-3,4,5-trihydroxyoxane-2-carboxylic acid

7.2.4. Number of Linked Drugs

The ADCs disclosed herein comprise drug molecules linked to antibody moieties in various stoichiometric molar ratios depending on the configuration of the antibody and, at least in part, the method used to effect conjugation.

The terms “drug load” or “drug loading” refer to the number of drug molecules per antibody in an individual ADC molecule. The number of TOP1i drugs linked to an anti-c-Met ADC can vary and will be limited by the number of available attachments sites on the anti-c-Met antibody. As contemplated for the anti-c-Met ADCs of the invention, the linker will link a single TOP1i drug to the antibody an anti-c-Met ADC. As long as the anti-c-Met ADC does not exhibit unacceptable levels of aggregation under the conditions of use and/or storage, anti-c-Met ADCs (i.e., structural formula (III)) having an n of up to 10 are contemplated. In some embodiments, the anti-c-Met ADCs have an n of in the range of from 1-10. In some embodiments, the anti-c-Met ADCs have an n selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, n is 2, 4, 6, 8, or 10. In embodiments, n is 6. In embodiments, the drug loading may comprise 1 drug molecule, 2 drug molecules, 3 drug molecules, 4 drug molecules, 5 drug molecules, 6 drug molecules, 7 drug molecules, 8 drug molecules, 9 drug molecules, or 10 drug molecules.

7.2.5. Antibody AbA

AbA is a humanized version of mouse monoclonal antibody 224G11, which was first disclosed and embodied in U.S. Pat. No. 8,329,173. The murine antibody m224G11 has a variable heavy domain set forth as SEQ ID NO: 13 and a variable light domain set forth as SEQ ID NO: 14: m224G11 Heavy Chain Variable Domain (CDRs underlined, and set forth as SEQ ID NOs: 15, 16, and 17)

(SEQ ID NO: 13) EVQLQQSGPELVKPGASVKISCKTS GYIFTAYT MHWVRQSLGESLDWIGG IKPNNGLA NYNQKFKGKATLTVDKSSSTAYMDLRSLTSEDSAVYYC ARSE ITTEFDY WGQGTALTVSS m224G11 Light Chain Variable Domain (CDRs underlined, and set forth as SEQ ID NOs: 18, 19, and 20):

(SEQ ID NO: 14) DIVLTQSPASLAVSLGQRATISCRAS ESVDSYANSF MHWYQQKPGQPPKL LIY RAS NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC QQSKEDPL T FGSGTKLEMK

AbA is a humanized recombinant IgG1κ (disclosed as 224G11 [TH7 Hz3] in U.S. Pat. No. 8,741,290) that targets a unique epitope of c-Met located within the immunoglobulin-plexin-transcription factor homology (IPT) domain 1, resulting in blockade of both HGF-dependent and HGF-independent c-Met signaling.

As defined under the IMGT nomenclature, the CDR sequences of AbA comprise the following sequences:

CDR-H1: (SEQ ID NO: 1) GYIFTAYT CDR-H2: (SEQ ID NO: 2) IKPNNGLA CDR-H3: (SEQ ID NO: 3) ARSEITTEFDY CDR-L1: (SEQ ID NO: 4) ESVDSYANSF CDR-L2: (SEQ ID NO: 5) RAS CDR-L3: (SEQ ID NO: 6) QQSKEDPLT

In some embodiments, the anti-c-Met antibodies composing an ADC of this disclosure comprise a CDR-H1 having the amino acid sequence shown as SEQ ID NO: 1, a CDR-H2 having the amino acid sequence shown as SEQ ID NO: 2; a CDR-H3 having the amino acid sequence shown as SEQ ID NO: 3, a CDR-L1 having the amino acid sequence shown as SEQ ID NO: 4, a CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5; and a CDR-L3 having the amino acid sequence shown as SEQ ID NO: 6.

In some embodiments, the anti-c-Met antibodies composing an ADC of this disclosure comprise a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 7:

(SEQ ID NO: 7) QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMGW IKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSE ITTEFDYWGQGTLVTVSS; and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 8:

(SEQ ID NO: 8) DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPKL LIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPL TFGGGTKVEIK.

In some embodiments, the anti-c-Met antibodies composing an ADC of this disclosure comprise a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 (constant regions are bold; CDRs are underlined (disclosed as SEQ ID NOS: 1-3, respectively, in order of appearance)):

QVQLVQSGAE VKKPGASVKV SCKASGYIFT AYTMHWVRQA PGQGLEWMGW 050 IKPNNGLANY AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARSE 100 ITTEFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 150 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 200 CNVNHKPSNT KVDKRVEPKS CDCHCPPCPA PELLGGPSVF LFPPKPKDTL 250 MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 300 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 350 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 400 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG 445 (full-length sequence disclosed as SEQ ID NO: 9) and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10 (CDR sequences disclosed as SEQ ID NOS: 4-6, respectively, in order of appearance):

DIVMTQSPDS LAVSLGERAT INCKSSESVD SYANSFLHWY QQKPGQPPKL 050 LIYRASTRES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQSKEDPL 100 TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV 150 QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV 200 THQGLSSPVT KSFNRGEC 218 (full-length sequence disclosed as SEQ ID NO: 10).

In embodiments, an antibody of the present disclosure comprises a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

In one embodiment, the heavy chain of an anti-c-Met antibody composing an ADC of this disclosure is encoded by the following nucleotide sequence (full-length sequence disclosed as SEQ ID NO: 21):

(SEQ ID NO: 21) ATGGGATGGTCTTGGATCTTTCTGCTGTTTCTGTCTGGTACTGCTGGTGT GCTGAGCcaggtccagctggtgcaatccggcgcagaggtgaagaagccag gcgcttccgtgaaggtgagctgtaaggcctctggctacatcttcacagca tacaccatgcactgggtgaggcaagctcctgggcagggactggagtggat gggatggattaaacccaacaatgggctggccaactacgcccagaaattcc agggtagggtcactatgacaagggataccagcatcagcaccgcatatatg gagctgagcaggctgaggtctgacgacactgctgtctattattgcgccag gagcgaaattacaacagaattcgattactgggggcagggcaccctggtga ccgtgtcctctgccagcaccaagggcccaagcgtgttccccctggccccc agcagcaagagcaccagcggcggcacagccgccctgggctgcctggtgaa ggactacttccccgagcccgtgaccgtgtcctggaacagcggagccctca cttctggagttcataccttcccagcagtattgcagagcagtggcctgtat tcactgtcttccgtcgtaacagttccatcctccagcctcgggacacagac ttacatttgtaacgtgaatcacaagcctagcaacaccaaggtcgacaaga gagttgaaccaaagagttgtgattgccactgtcctccctgcccagctcct gagctgcttggcggtcccagtgtcttcttgtttccccctaaacccaaaga caccctgatgatctcaaggactcccgaggtgacatgcgtggtggtggatg tgtctcatgaggacccagaggtgaagttcaactggtacgtggacggcgtg gaggtgcacaacgccaagaccaagcccagagaggagcagtacaacagcac ctacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacg gcaaggagtacaagtgtaaggtgtccaacaaggccctgccagccccaatc gaaaagaccatcagcaaggccaagggccagccaagagagccccaggtgta caccctgccacccagcagggaggagatgaccaagaaccaggtgtccctga cctgtctggtgaagggcttctacccaagcgacatcgccgtggagtgggag agcaacggccagcccgagaacaactacaagaccacccccccagtgctgga cagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagagca gatggcagcagggcaacgtgttcagctgctccgtgatgcacgaggccctg cacaaccactacacccagaagagcctgagcctgtccccaggctga Secretion signal peptide in bold CAPITAL letters; includes final stop codon (TGA); constant region is bold; CDRs are underlined (CDR sequences disclosed as SEQ ID NOS: 22-24, respectively, in order of appearance)

In one embodiment, the light chain of an anti-c-Met antibody composing an ADC of this disclosure is encoded by the following nucleotide sequence (full-length sequence disclosed as SEQ ID NO: 25):

(SEQ ID NO: 25) ATGGAAACTGATACACTGCTGCTGTGGGTCCTGCTGCTGTGGGTCCCTGG AAGCACAGGGgacattgtgatgacccagtctcccgatagcctggccgtgt ccctgggcgagagggctaccatcaactgtaaaagctccgaatctgtggac tcttacgcaaacagctttctgcactggtatcagcaaaagccaggccaacc tccaaagctgctgatttacagggcttctaccagggagagcggcgtgcccg ataggttcagcggatctggcagcggcaccgactttacactgaccatctcc agcctgcaggccgaagatgtggcagtctattactgccagcagtccaagga ggaccccctgactttcgggggtggtactaaagtggagatcaagcgtacgg tggccgctcccagcgtgttcatcttccccccaagcgacgagcagctgaag agcggcaccgccagcgtggtgtgtctgctgaacaacttctaccccaggga ggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagcc aggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagc agcaccctgaccctgagcaaggccgactacgagaagcacaaggtgtacgc ctgtgaggtgacccaccagggcctgtccagccccgtgaccaagagcttca acaggggcgagtgctga Secretion signal peptide in bold CAPITAL letters; includes final stop codon (TGA); constant region is bold; CDRs are underlined (CDR sequences disclosed as SEQ ID NOS: 26-28, respectively, in order of appearance).

7.2.6. Exemplary Anti-c-Met ADCs

In particular embodiments, anti-c-Met ADCs of the invention comprise an anti-c-Met antibody comprising six complementarity determining regions (CDRs) corresponding to the CDRs of antibody AbA, which is conjugated to a TOP1i drug through a cleavable valine alanine (va) linker. In certain embodiments, the linker comprises a bromoacetamide functional group for conjugation to a sulfhydryl of a reduced cysteine from the anti-c-Met antibody of the ADC. In other embodiments, the linker comprises a maleimide functional group for conjugation to a sulfhydryl of a reduced cysteine from the anti-c-Met antibody of the ADC. In some embodiments, the linker further comprises a self-immolating spacer, preferably p-aminobenzylcarbonyl (PABC) or an analogue thereof.

In embodiments, an anti-c-Met ADC comprises an anti-c-Met antibody comprising six complementarity determining regions (CDRs) corresponding to the CDRs of antibody AbA, which is conjugated to linker drug LD1 (structural formula (V)) via a linkage formed with a sulfhydryl group of a cysteine residue of the anti-c-Met antibody. In some embodiments, the anti-c-Met antibody comprises the variable heavy (VH) chain and variable light (VL) chain region sequences of antibody AbA. In some embodiments, the anti-c-Met antibody comprises the heavy chain (HC) and light chain (LC) sequences of antibody AbA.

In embodiments, an anti-c-Met ADC has the following structural formula (VIII):

wherein n is an integer from 1-10, and wherein Ab is an IgG1 anti-c-Met antibody comprising a heavy chain CDR1 shown as SEQ ID NO: 1, a heavy chain CDR2 shown as SEQ ID NO: 2, a heavy chain CDR3 shown as SEQ ID NO: 3, a light chain CDR1 shown as SEQ ID NO: 4, a light chain CDR2 shown as SEQ ID NO: 5, and a light chain CDR3 shown as SEQ ID NO: 6. In embodiments, the antibody Ab is an IgG1 anti-c-Met antibody comprising a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 8. In embodiments, the antibody Ab is an anti-c-Met antibody comprising a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10. In embodiments, conjugation of the linker-drug to the antibody is via a linkage formed with a sulfhydryl group of a cysteine residue of the antibody. In embodiments, n has a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, n has a value of 2, 4, 6, 8, or 10. In embodiments, n is 6. In an embodiment, anti-c-Met ADC of structural formula (VIII) comprises an antibody Ab having a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10 and n has a value of 2, 4, 6, 8, or 10. In an embodiment, anti-c-Met ADC of structural formula (VIII) comprises an antibody Ab having a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10 and n has a value of 6.

In certain embodiments, an anti-c-Met ADC comprises an anti-c-Met antibody comprising six complementarity determining regions (CDRs) corresponding to the CDRs of antibody AbA, which is conjugated to linker drug LD2 (structural formula (VI)) via a linkage formed with a sulfhydryl group of a cysteine residue of the anti-c-Met antibody. In some embodiments, the anti-c-Met antibody comprises the variable heavy (VH) chain and variable light (VL) chain region sequences of antibody AbA. In some embodiments, the anti-c-Met antibody comprises the heavy chain (HC) and light chain (LC) sequences of antibody AbA.

In some embodiments, an anti-c-Met ADC has the following structural formula (IX):

wherein n is an integer from 1-10, and wherein Ab is an IgG1 anti-c-Met antibody comprising a heavy chain CDR1 shown as SEQ ID NO: 1, a heavy chain CDR2 shown as SEQ ID NO: 2, a heavy chain CDR3 shown as SEQ ID NO: 3, a light chain CDR1 shown as SEQ ID NO: 4, a light chain CDR2 shown as SEQ ID NO: 5, and a light chain CDR3 shown as SEQ ID NO: 6. In embodiments, the antibody Ab is an IgG1 anti-c-Met antibody comprising a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 8. In embodiments, the antibody Ab is an anti-c-Met antibody comprising a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10. In embodiments, conjugation of the linker-drug to the antibody is via a linkage formed with a sulfhydryl group of a cysteine residue of the antibody. In embodiments, n has a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, n has a value of 2, 4, 6, 8, or 10. In embodiments, n is 6. In an embodiment, anti-c-Met ADC of structural formula (IX) comprises an antibody Ab having a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10, and n has a value of 2, 4, 6, 8, or 10. In an embodiment, anti-c-Met ADC of structural formula (IX) comprises an antibody Ab having a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10 and n has a value of 6.

In certain embodiments, an anti-c-Met ADC comprises an anti-c-Met antibody comprising six complementarity determining regions (CDRs) corresponding to the CDRs of antibody AbA, which is conjugated to linker drug LD3 (structural formula (VII)) via a linkage formed with a sulfhydryl group of a cysteine residue of the anti-c-Met antibody. In some embodiments, the anti-c-Met antibody comprises the variable heavy (VH) chain and variable light (VL) chain region sequences of antibody AbA. In some embodiments, the anti-c-Met antibody comprises the heavy chain (HC) and light chain (LC) sequences of antibody AbA.

In some embodiments, an anti-c-Met ADC has the following structural formula (X):

wherein n is an integer from 1-10, and wherein Ab is an IgG1 anti-c-Met antibody comprising a heavy chain CDR1 shown as SEQ ID NO: 1, a heavy chain CDR2 shown as SEQ ID NO: 2, a heavy chain CDR3 shown as SEQ ID NO: 3, a light chain CDR1 shown as SEQ ID NO: 4, a light chain CDR2 shown as SEQ ID NO: 5, and a light chain CDR3 shown as SEQ ID NO: 6. In embodiments, the antibody Ab is an IgG1 anti-c-Met antibody comprising a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 8. In embodiments, the antibody Ab is an anti-c-Met antibody comprising a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10. In embodiments, conjugation of the linker-drug to the antibody is via a linkage formed with a sulfhydryl group of a cysteine residue of the antibody. In embodiments, n has a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, n has a value of 2, 4, 6, 8, or 10. In embodiments, n is 6. In an embodiment, anti-c-Met ADC of structural formula (X) comprises an antibody Ab having a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10 and n has a value of 2, 4, 6, 8, or 10. In an embodiment, anti-c-Met ADC of structural formula (X) comprises an antibody Ab having a heavy chain comprising the amino acid sequence shown as SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown as SEQ ID NO: 10 and n has a value of 6.

The ADCs of this disclosure may be provided as a composition suitable for administration to a subject. In some embodiments, the ADC composition is a pharmaceutical composition, comprising an ADC of this disclosure and a pharmaceutically acceptable carrier. A given formulation of the ADCs disclosed herein may comprise a distribution of antibodies having differing drug loading, i.e., differing values of n.

7.3. Methods of Use

In embodiments, the methods described herein involve treating patients who have non-squamous NSCLC with the anti-c-Met ADCs of the invention.

8. EXAMPLES

The following Examples, which highlight certain features and properties of the exemplary embodiments of the antibodies and binding fragments described herein are provided for purposes of illustration.

Example 1 (2S)-2-(2-bromoacetamido)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

Example 1A tert-butyl (3-(methoxy(methyl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamate

To a solution of 3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylic acid (4.9 g), N,O-dimethylhydroxylamine hydrochloride (2.2 g) and N,N-diisopropylethylamine (11.30 mL) in dichloromethane (10 mL) was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (8.61 g) in portions at 10° C. The reaction mixture was stirred at 20° C. for 12 hours. Two additional reactions were set up and allowed to stir at 20° C. for 12 hours as described. All three reactions were combined. The reaction was diluted with dichloromethane (200 mL) and added to 1 N aqueous HCl (50 mL). The precipitate formed was filtered and the filtrate was allowed to separate. The organic layer was washed with saturated aqueous sodium bicarbonate solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel, eluting with 1-50% ethyl acetate in petroleum ether to give the title compound. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.97 (br s, 1H), 3.66 (s, 3H), 3.18 (s, 3H), 2.34 (s, 6H), 1.45 (s, 9H). MS (ESI+) m/z 271.2 (M+H)⁺.

Example 1B tert-butyl (3-(benzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)carbamate

To a solution of 5-bromobenzo[d][1,3]dioxole (8.83 g) in tetrahydrofuran (100 mL) was added n-butyllithium (17.57 mL, 2.5 M in hexane) slowly at −65° C. under nitrogen gas. The mixture was stirred at −65° C. for 30 minutes. A solution of Example 1A (4.75 g) in tetrahydrofuran (40 mL) was added slowly. The mixture was stirred at −65° C. for 3 hours. Three additional reactions were set up and allowed to stir at −65° C. for 3 hours. All four reactions were combined. The mixture was quenched with saturated aqueous ammonium chloride solution (500 mL) and extracted with ethyl acetate (3×500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel, eluting with 1-50% ethyl acetate in petroleum ether to give the title compound. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.63 (dd, 1H), 7.45 (d, 1H), 6.82 (d, 1H), 6.03 (s, 2H), 5.04 (br s, 1H), 2.50 (s, 6H), 1.46 (s, 9H). MS (ESI+) m/z 354.2 (M+Na)⁺.

Example 1C N-(3-(benzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

Step 1: To a solution of Example 1B (6.2 g) in dichloromethane (62 mL) was added trifluoroacetic acid (62 mL) slowly at 0° C. The reaction was stirred at 25° C. for 4 hours. Two additional reactions were set up and stirred at 25° C. for 4 hours. Each mixture was concentrated under reduced pressure. Each residue was used in the next step without further purification.

Step 2: To a solution of crude product above in dichloromethane (62 mL) was added N,N-diisopropylethylamine (16.34 mL) and trifluoroacetic anhydride (3.96 mL) dropwise at 0° C. The mixture was stirred at 25° C. for 2 hours. Two additional reactions were set up as described and stirred at 25° C. for 2 hours. All three reactions were combined and poured into water (200 mL), extracted with dichloromethane (2×200 mL). The organic layer was washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with 25% ethyl acetate in petroleum ether to give the title compound. ¹H NMR (501 MHz, CDCl₃) δ ppm 7.61 (dd, 1H), 7.43 (d, 1H), 7.00 (s, 1H), 6.85 (d, 1H), 6.05 (s, 2H), 2.62 (s, 6H). MS (ESI+) m/z 328.2 (M+H)⁺.

Example 1D 2,2,2-trifluoro-N-(3-(6-nitrobenzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)acetamide

To a solution of Example 1C (4.3 g) in acetic anhydride (25 mL) was added copper(II) nitrate trihydrate (4.76 g) in portions at 0° C. The mixture was stirred at 0° C. for 3 hours. Three additional reactions were set up and stirred at 0° C. for 3 hours as described. All four reactions were combined. The mixture was poured into water (50 mL) and extracted with ethyl acetate (5×100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with 75% ethyl acetate in petroleum ether to give the title compound. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.61 (s, 1H), 6.77 (br s, 1H), 6.64 (s, 1H), 6.21 (s, 2H), 2.43 (s, 6H). MS (APCI+) m/z 373.1 (M+H)⁺.

Example 1E N-(3-(6-aminobenzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

To a solution of Example 1D (4 g) in ethanol (40 mL) and water (8 mL) was added iron (5.4 g) and ammonium chloride (5.17 g) under nitrogen. The mixture was stirred at 100° C. for 3 hours. Three additional reactions were set up and stirred at 100° C. for 3 hours as described. After cooling to ambient temperature, all four reactions were combined. The mixture was poured into water (1 L) and extracted with ethyl acetate (5×500 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with 10-75% ethyl acetate in petroleum ether to give the title compound. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.22 (s, 1H), 6.70 (s, 1H), 6.50 (s, 2H), 6.14 (s, 1H), 5.92 (s, 2H), 2.63 (s, 6H). MS (ESI+) m/z 343.2 (M+H)⁺.

Example 1F (S)—N-(3-(7-ethyl-7-hydroxy-8,11-di oxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

To a suspension of Example 1E (3.5 g) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2.69 g) in toluene (140 mL) was added para-toluenesulfonic acid monohydrate (1.945 g). The mixture was stirred at 115° C. for 12 hours. Three additional reactions were set up and stirred at 115° C. for 12 hours as described. After cooling to ambient temperature, all four reactions were combined. The mixture was filtered and the solid collected was triturated with acetonitrile (200 mL) to give the title compound. 1H NMR (400 MHz, dimethyl sulfoxide—d₆) δ ppm 10.28 (s, 1H), 7.64 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.47 (s, 1H), 6.30 (dd, 2H), 5.42 (s, 2H), 5.36 (s, 2H), 2.87 (s, 6H), 1.94-1.77 (m, 2H), 0.88 (t, 3H). MS (ESI+) m/z 570.3 (M+H)⁺.

Example 1G: (7S)-14-(3-aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-8,11(7H,13H)-dione

To a solution of Example 1F (3 g) in methanol (30 mL) was added HCl (60 mL, 4 M in methanol). The mixture was stirred at 65° C. for 4 hours. Four additional reactions were set up and stirred at 65° C. for 4 hours as described above. After cooling to ambient temperature, all five reactions were combined. The mixture was concentrated under reduced pressure and the residue was triturated with methanol (200 mL) to give the title compound. 1H NMR (400 MHz, dimethyl sulfoxide—d₆) δ ppm 9.23 (s, 3H), 7.54 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.30 (d, 2H), 5.41 (s, 2H), 5.39-5.26 (m, 2H), 2.79 (s, 6H), 1.87 (hept, 2H), 0.88 (t, 3H). MS (ESI+) m/z 474.3 (M+H)⁺.

Example 1H tert-butyl ((S)-1-(((S)-1-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a suspension of (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propanoic acid (2.49 g), 2-hydroxypyridine 1-oxide (1.31 g), and N¹-((ethylimino)methylene)-N³,N³-dimethylpropane-1,3-diamine hydrochloride (2.26 g) in acetonitrile (40 mL) was added 2,6-lutidine (2.74 mL). The mixture was stirred at ambient temperature for 30 minutes. In a separate flask, Example 1G (4 g) and 2,6-lutidine (2.74 mL) were combined in N,N-dimethylformamide (40 mL) and the above solution was added. The mixture was stirred at ambient temperature overnight. The mixture was concentrated under reduced pressure and the residue was dissolved in dichloromethane (300 mL), washed with saturated aqueous ammonium chloride solution (100 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with 0-10% methanol in dichloromethane to give the title compound. ¹H NMR (400 MHz, dimethyl sulfoxide—d₆) δ ppm 8.65 (s, 1H), 7.89 (d, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 7.22 (s, 1H), 6.77 (d, 1H), 6.46 (s, 1H), 6.29 (d, 2H), 5.41 (s, 2H), 5.32 (s, 2H), 4.29 (q, 1H), 3.87-3.77 (m, 1H), 2.76 (s, 6H), 1.99 (q, 1H), 1.92-1.81 (m, 2H), 1.41 (s, 9H), 1.25 (d, 3H), 0.92-0.80 (m, 9H). MS (ESI+) m/z 744.4 (M+H)⁺.

Example 1I (S)-2-amino-N—((S)-1-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

Example 1H (5.5 g) was treated with trifluoracetic acid (30 mL) at ambient temperature for 30 minutes. The mixture was concentrated under reduced pressure and the residue was dissolved in 50% acetonitrile in water (200 mL). The solution was lyophilized to give the title compound. ¹H NMR (600 MHz, dimethyl sulfoxide—d₆) δ ppm 8.80 (s, 1H), 8.59 (d, 1H), 8.10 (d, 3H), 7.60 (s, 1H), 7.48 (s, 1H), 7.23 (s, 1H), 6.33-6.26 (m, 2H), 5.41 (d, 2H), 5.35-5.23 (m, 2H), 4.35 (p, 1H), 3.66-3.63 (m, 1H), 2.77 (s, 6H), 2.17-2.06 (m, 1H), 1.91-1.83 (m, 2H), 1.31 (d, 3H), 1.01-0.96 (dd, 6H), 0.89 (t, 3H). MS (ESI+) m/z 644.4 (M+H)⁺.

Example 1J (2S)-2-(2-bromoacetamido)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

To a solution of 2-bromoacetic acid (1.435 g) in N,N-dimethylformamide (26 mL) was added ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.55 g). The mixture was stirred at ambient temperature for 10 minutes. In a separate flask, Example 1I (4.5 g) and 2,6-lutidine (3.61 mL) were combined in N,N-dimethylformamide (26 mL) and the above solution was added. The mixture was stirred at ambient temperature for 30 minutes. The mixture was acidified with trifluoroacetic acid (4 mL) and purified by reversed-phase HPLC on a CombiFlash® Teledyne Isco system using a Luna column (250×50 mm, 10 mm), eluting with 5-75% acetonitrile in water containing 0.1% trifluoroacetic acid over 30 minutes to give the title compound after lyophilization. ¹H NMR (600 MHz, dimethyl sulfoxide—d₆) δ ppm 8.57 (s, 1H), 8.32 (d, 1H), 8.16 (d, 1H), 7.67 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.30 (dd, 2H), 5.42 (s, 2H), 5.38 (d, 2H), 4.28-4.19 (m, 2H), 4.03-3.91 (m, 2H), 2.76 (s, 6H), 2.02 (h, 1H), 1.86 (ddp, 2H), 1.25 (d, 3H), 0.93-0.84 (m, 9H). MS (ESI+) m/z 764.46 (M+H)⁺.

Example 2 (2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

A solution of Example 1I (2 g) in N,N-dimethylformamide (54 mL) was added 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.7 g) followed by N,N-diisopropylethylamine (2.3 mL). The mixture was stirred at ambient temperature for 30 minutes. The reaction was quenched with trifluoroacetic acid (2 mL) and purified by reversed-phase HPLC on a Gilson PLC 2020 system using a Luna column (250×50 mm, 10 mm), eluting with 5-75% acetonitrile in water containing 0.1% trifluoroacetic acid over 30 minutes to provide the title compound after lyophilization. ¹H NMR (500 MHz, dimethyl sulfoxide—d₆) δ ppm 8.50 (s, 1H), 8.29 (d, 1H), 8.13 (d, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.23 (s, 1H), 7.12 (s, 2H), 6.30 (d, 2H), 5.42 (s, 2H), 5.36 (d, 2H), 4.24 (p, 1H), 4.20-4.14 (m, 3H), 2.75 (s, 6H), 2.01 (h, 1H), 1.86 (dp, 2H), 1.26 (d, 3H), 0.93-0.83 (m, 9H). MS (ESI+) m/z 781.14 (M+H)⁺.

Example 3 (2S,3S,4R,5R,6S)-6-[2-(5-{[(2S)-2-({(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-3-methylbutanoyl}amino)propanoyl]amino}-2-{{3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3%4′: 6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}carbamoyl)oxy]methyl}phenyl)ethyl]-3,4,5-trihydroxyoxane-2-carboxylic acid

Example 3A (2S,3S,4R,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)carbamoyl)oxy)methyl)phenethyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

To a mixture of (2S,3S,4R,5S,6S)-2-(5-((S)-2-((S)-2-4((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenethyl)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (prepared as described in WO2016094509, 2.99 g), Example 1G (1.35 g) and 1-hydroxy-7-azabenzotriazole (0.361 g) in N,N-dimethylformamide (26.5 mL) was added N,N-diisopropylethylamine (1.39 mL). The mixture was stirred at ambient temperature for 4 hours. The mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel, eluting with 0-8% methanol in dichloromethane to give the title compound. ¹H NMR (501 MHz, CDCl₃) δ ppm 8.43 (br s, 1H), 7.74 (d, 2H), 7.61 (br s, 1H), 7.57-7.46 (d, 4H), 7.43 (s, 1H), 7.38 (t, 3H), 7.33-7.27 (m, 3H), 6.43 (br s, 1H), 6.17 (s, 2H), 5.89 (br s, 1H), 5.72 (d, 1H), 5.37-5.02 (m, 8H), 4.94 (t, 1H), 4.67-4.56 (m, 1H), 4.53-4.44 (t, 2H), 4.19 (br s, 1H), 4.00 (d, 2H), 3.77 (s, 3H), 3.48 (dt, 1H), 2.94 (d, 1H), 2.88-2.66 (m, 6H), 2.16 (br s, 1H), 2.08-1.97 (m, 9H), 1.94-1.80 (ddt, 4H), 1.45 (d, 3H), 1.02 (t, 3H), 0.98-0.90 (m, 6H). MS (ESI+) m/z 1359.5 (M+H)⁺.

Example 3B (2S,3S,4R,5R,6S)-6-(5-(S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-((((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)carbamoyl)oxy)methyl)phenethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

To a solution of Example 3A (3.2 g) in methanol (24 mL) and tetrahydrofuran (24 mL) was added lithium hydroxide monohydrate (846 mg) in water (24 mL). The mixture was stirred at ambient temperature for 30 minutes. The reaction was quenched with trifluoracetic acid (3 mL) and extracted with heptane (5×30 mL). The aqueous layer was purified by reversed-phase HPLC on a CombiFlash® Teledyne Isco system using a Luna column (250×50 mm, 10 mm), eluting with 5-75% acetonitrile in water containing 0.1% trifluoroacetic acid over 30 minutes to provide the title compound after lyophilization. ¹H NMR (400 MHz, dimethyl sulfoxide—d₆) δ ppm 10.03 (s, 1H), 8.62 (d, 1H), 8.02 (br s, 4H), 7.55 (s, 1H), 7.43 (s, 1H), 7.41 (d, 2H), 7.35 (s, 1H), 7.22 (d, 1H), 7.16 (s, 1H), 6.40 (br s, 1H), 6.23 (s, 2H), 5.35 (s, 2H), 5.27 (s, 2H), 4.97 (s, 2H), 4.43 (p, 1H), 3.60-3.47 (m, 3H), 3.15-3.01 (m, 3H), 2.90 (t, 1H), 2.64 (s, 6H), 2.00 (tq, 2H), 1.79 (hept, 2H), 1.60-1.46 (m, 1H), 1.29 (d, 3H), 0.89 (dd, 6H), 0.80 (t, 3H). MS (ESI+) m/z 997.3 (M+H)⁺.

Example 3C

(2S,3S,4R,5R,6S)-6-[2-(5-{[(2S)-2-({(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]-3-methylbutanoyl}amino)propanoyl]amino}-2-{[({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}carbamoyl)oxy]methyl}phenyl)ethyl]-3,4,5-trihydroxyoxane-2-carboxylic acid

A solution of Example 3B (1.02 g) in N,N-dimethylformamide (16 mL) was added 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.28 g) followed by N,N-diisopropylethylamine (0.8 mL). The mixture was stirred at ambient temperature for 30 minutes. The reaction was quenched with acetic acid (0.8 mL) and purified by reversed-phase HPLC on a Gilson PLC 2020 system using a Luna column (250×50 mm, 10 mm), eluting with 5-75% acetonitrile in water containing 0.1% trifluoroacetic acid over 30 minutes to provide the title compound after lyophilization. 1H NMR (501 MHz, dimethyl sulfoxide—d₆) δ ppm 9.90 (s, 1H), 8.29-8.25 (m, 2H), 8.11 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.47 (d, 1H), 7.42 (s, 1H), 7.27 (d, 1H), 7.23 (s, 1H), 7.08 (s, 2H), 6.30 (s, 2H), 5.42 (s, 2H), 5.36 (s, 2H), 5.03 (s, 2H), 4.38 (p, 1H), 4.22 (dd, 1H), 4.13 (s, 2H), 3.58 (d, 2H), 3.15 (d, 4H), 2.97 (t, 1H), 2.71 (s, 6H), 2.10-1.93 (m, 2H), 1.86 (dp, 2H), 1.64-1.52 (m, 1H), 1.31 (d, 3H), 0.91-0.79 (m, 9H). MS (ESI+) m/z 1134.3 (M+H)⁺.

Example 4: Cell Proliferation

The effect of TOP1i drugs on cell viability was assessed by monitoring ATP presence for metabolically active cells. Proliferation of Calu-6 cells (lung adenocarcinoma) and A375 cells (malignant melanoma) were quantified using CellTiter-Glo® luminescence. As shown in FIG. 2A and in Table 1, the compound of Formula I (Example 1G) exhibited sub-nanomolar potency for reducing viability of proliferating Calu-6 cells (0.57 nM, FIG. 2A) and A375 (0.50 nM, FIG. 2B). This sub-nanomolar potency improved over performance of the TOP1i drug SN-38 in the same assay, which demonstrated an EC₅₀ of 6.58 nM (Calu-6) and 2.30 nM (A375).

TABLE 1 TOP1i Compound Potency EC₅₀ (nM) Compound Calu-6 A375 SN-38 6.58 2.30 Example 1G 0.57 0.50

Example 5: Preparation and Purification of AbA

AbA was expressed in Chinese hamster ovary (CHO) cells with the heavy and light chain sequences of SEQ ID NOS: 9 and 10, respectively. AbA was isolated and purified prior to conjugation.

Example 6: Procedure for Conjugation (ADC-1) Conjugation Step 1

Prior to reduction, a solution of anti-c-Met antibody AbA was incubated at 4° C. for a minimum of 12 hours. A 0.5 M ethylenediaminetetraacetic acid disodium salt solution (EDTA, Sigma Aldrich) was then added, for a final concentration of 5 mM. Tris (2-carboxyethyl) phosphine (TCEP, 100 mM, 2.67 molar equivalents, Bond Breaker™, Thermo Scientific™) was added to the solution (approximately 10 mg/mL in 1×DPBS (Dulbecco's phosphate-buffered saline) with gentle stir plate mixing (225 rpm) for 5 minutes. Following refrigerator incubation at 4° C. for 17 hours, the solution was brought to 22° C. on a stir plate mixing at 225 rpm. A 10% v/v N,N-dimethylacetamide (DMA, Sigma Aldrich) co-solvent was then added, followed by 20% v/v of prepared 1 M boric acid, pH 8.0 (Sigma Aldrich) and then 6 mol equivalents of 50 mM linker drug LD1 (structural formula V, Example 1J) in N,N-dimethylacetamide, dropwise. Mixing was discontinued after 20 minutes and the solution was incubated at 22° C. for 3.3 hours. Following incubation, 2 mol equivalents of 50 mM N-Acetyl-L-cysteine (NAC, Sigma Aldrich) prepared in 1×DPBS, were added to the solution, and gently mixed by manually agitating the vessel. Following a 1-hour incubation at 22° C., the solution was desalted using a 691 mL G25 Fine Desalting (Cytiva™) Column, into 1×DPBS.

Conjugation Step 2

Prior to reduction, a solution of anti-c-Met antibody AbA was incubated at 4° C. for a minimum of 12 hours. A 0.5 M ethylenediaminetetraacetic acid disodium salt solution (EDTA, Sigma Aldrich) was then added, for a final concentration of 5 mM. Tris (2-carboxyethyl) phosphine (TCEP, 100 mM, 1.00 mol eq, Bond Breaker™, Thermo Scientiflc™) was added to a solution of antibody (approximately 4 mg/mL in 1×DPBS) with gentle stir plate mixing (225 rpm) for 5 minutes. Following refrigerator incubation at 4° C. for 17 hours, the solution was brought to 22° C. on a stir plate mixing at 225 rpm. A 10% v/v N,N-dimethylacetamide (DMA, Sigma Aldrich) co-solvent was then added, followed by 20% v/v 1 M boric acid, pH 8.0 (Sigma Aldrich) and then 4 molar equivalents of 50 mM linker drug LD1 (structural formula V, Example 1J) in N,N-dimethylacetamide, dropwise. Mixing was discontinued after 20 minutes and the solution was incubated at 22° C. for 2.1 hours. Following incubation, 2 molar equivalents of 10 mM N-acetyl-L-cysteine (NAC, Sigma Aldrich), prepared in 1×DPBS, were added to the solution, and gently mixed by manually agitating the vessel. Following a 5-hour incubation at 22° C., the solution was concentrated, and buffer exchanged via Ultrafiltration Diafiltration (UF/DF).

UF/DF Step

A Pellicon® 3 0.22 m2 Cassette (Millipore) was flushed with 2 L of sterile water and then 0.5 L of formulation buffer. Ultrafiltration (UF) was performed at 250 mL/minute feed flow rate, and a Transmembrane Pressure (TMP) of 12-13 psi, with a starting volume of 1.3 L. Final permeate volume was 1.1 L. Diafiltration (DF) was performed with 25 diavolumes (DV) of final formulation buffer, at the same feed flow rate and TMP as the UF step. Following DF, a second UF step was performed to further reduce ADC solution volume to 100 mL. The ADC solution was removed, and the cassette was twice rinsed and with 125 mL of formulation buffer which was then ultrafiltered to 20 mL each. These rinses were then added to the bulk ADC solution. The final solution was sterile filtered with a 0.22 μm filter, prior to characterization.

Example 7: Procedure for Conjugation (ADC-2)

Prior to reduction, a solution of anti-c-Met antibody AbA was incubated on wet ice for 35 minutes. A 0.5 M ethylenediaminetetraacetic acid disodium salt solution (EDTA, Sigma Aldrich) was then added, for a final concentration of 5 mM. Tris (2-carboxyethyl) phosphine (TCEP, 25 mM, 3.50 molar equivalent, Bond Breaker™, Thermo Scientific™) was added to the solution (approximately 10 mg/mL in 1×DPBS) and gently mixed by slowly inverting the tube several times. The solution was then placed on wet ice and placed in the 4° C. refrigerator for 24 hours. Following incubation, 10% v/v N,N-dimethylacetamide co-solvent was added followed by 10 molar equivalents of 10 mM linker drug LD2 (structural formula VI, Example 2) in N,N-dimethylacetamide (DMA, Sigma Aldrich) solution, and gently mixed by slowly inverting the tube several times. The solution was then placed on wet ice for 30 minutes and incubated at 22° C. for 90 minutes. Following incubation, 8 molar equivalents of 10 mM N-acetyl-L-cysteine (NAC, Sigma Aldrich) prepared in PBS were added to the solution, and gently mixed by slowly inverting the tube several times. After a 60-minute incubation at 22° C., the solution was desalted over a HiPrep™ 26/10 desalting column into 1×DPBS (Cytiva™). Desalted ADCs were then hydrolyzed by the addition of a prepared solution of 10% v/v 1 M boric acid, pH 8.0 (Sigma Aldrich) with a 22° C. incubation, for 96 hours. Hydrolyzed ADCs were purified through coupled affinity to desalting chromatography (2×1 mL HiTrap MabSelect SuRe, HiPrep™ 26/10 desalting, Cytiva™) into a final formulation of 1×DPBS. Collected fractions were pooled and sterile filtered through a 0.22 μm filter prior to characterization.

Example 8: Procedure for Conjugation (ADC-3)

Aqueous ethylenediaminetetraacetic acid disodium salt (EDTA, 0.5 M, 21.3 μL, Sigma Aldrich) was added to 5.337 mL of a solution of anti-c-Met antibody (AbA, 13.34 mg/mL in 1×DPBS, pH 7.4). Tris(2-carboxyethyl) phosphine (TCEP, 10 mM, 3.0 molar eq, 142.4 μL, Bond Breaker™, Thermo Scientific™) was added to the solution, gently stirred, and kept at 37° C. for 75 minutes. The solution was cooled to ambient temperature and linker-drug LD3 (structural formula VII, Example 3C) was added (10 eq., 527 μL of 10 mM solution of LD3 (Structural Formula VII, Example 3C in N,N-dimethylacetamide, 90% purity)). The conjugation was gently stirred and allowed to stand for 1.2 hours at ambient temperature. The ADC solution was purified by desalting. After desalting, the ADC solution was filtered through 0.22 μm filter and resulting sample stored at 4° C. The resultant ADC was hydrolyzed by adding 10% v/v 1.0 M borate buffer, pH 8.0 and incubated in a dark environment at ambient temperature for 72 hours. After hydrolysis, the resultant ADC solution was purified by adsorbing to a protein A resin column (MabSelect™ SuRe™ LX, GE Healthcare); washing with 4 column volumes of 1×DPBS, pH 7.4; eluting off resin with 5 column volumes of IgG Elution Buffer (Thermo Scientific™); and desalted into 1×DPBS, pH 7.4. After desalting, the ADC solution was filtered through a 0.22 μm filter and the resulting sample was stored at 4° C.

Example 9: Procedure for Conjugation (ADC-4)

Aqueous ethylenediaminetetraacetic acid disodium salt (EDTA, 0.5 M, 974 μL, Sigma Aldrich) was added to 243.3 mL of a purified solution of MSL109-C6v1 antibody (12.3 mg/mL in 20 mM Tris buffer, pH 7.4). MSL109-C6v1 is a monoclonal antibody that binds to CMV glycoprotein H. MSL109-C6v1 comprises a heavy chain set forth as SEQ ID NO: 11 and a light chain set forth as SEQ ID NO: 12 and is used as a non-targeting control antibody. Tris(2-carboxyethyl) phosphine (TCEP, 500 mM, 6.0 molar eq, 242.4 [IL, Bond Breaker™, Thermo Scientific™) was added to the solution of MSL109-C6v1 with 2 mM EDTA, and gently stirred and kept at 4° C. for 20 hours. Tris buffer at 4° C. (1.0 M, pH 8.0) was added to the solution (24.5 mL, 10% v/v). The linker-drug LD1 (Example 1J, (2S)-2-(2-bromoacetamido)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide) was added to the reduced antibody at pH 8.0 (10 equivalents, 22.5 mL of 10 mM solution of LD1, structural formula V, Example 1J, in N,N-dimethylacetamide, 90% purity). The conjugation was gently stirred and allowed to stand for 1.5 hours in an ambient temperature water bath. The conjugation was quenched with addition of 4 equivalents of an aqueous solution of N-acetyl-L-cysteine (NAC, Sigma Aldrich, 100 mM solution in 1×DPBS, 0.8 mL). The mixture was gently stirred and allowed to stand for 1 hour at ambient temperature. Tris buffer (15%, Sigma, 1.0 M, pH 7.4, 44 mL) was added to adjust the ADC solution to pH 7.5. The ADC solution was stored at 4° C. overnight. The ADC solution was concentrated, and buffer exchanged via Ultrafiltration Diafiltration (Pellicon® 3 0.22 m2 Cassette (Millipore)). The final ADC solution was filtered through a 0.22 μm filter and the resulting ADC sample stored at 4° C. Final volume of ADC was 138 mL.

Example 10: DAR (Drug Antibody Ratio) Determination

DAR was determined by LC-MS. LC-MS analysis was performed using an Agilent 1100 HPLC system interfaced to an Agilent LC/MSD TOF 6220 ESI mass spectrometer. The ADC was reduced with 5 mM (final concentration) Bond-Breaker™ TCEP solution (Thermo Scientific™ Rockford, IL), loaded onto a Protein Microtrap (Michrom Bioresources, Auburn, CA) desalting cartridge, and eluted with a gradient of 10% B to 75% B in 0.2 minutes at ambient temperature. Mobile phase A was H₂O with 0.1% formic acid (FA), mobile phase B was acetonitrile with 0.1% FA, and the flow rate was 0.2 mL/minute. Electrospray-ionization time-of-flight mass spectra of the co-eluting light and heavy chains were acquired using Agilent MassHunter™ acquisition software. The extracted intensity vs. m/z spectrum was deconvoluted using the Maximum Entropy feature of MassHunter™ software to determine the mass of each reduced antibody fragment. DAR was calculated from the deconvoluted spectrum by summing intensities of the naked and modified peaks for the light chain and heavy chain, normalized by multiplying intensity by the number of drugs attached. The summed, normalized intensities were divided by the sum of the intensities, and the summing results for two light chains and two heavy chains produced a final average DAR value for the full ADC. Thiosuccinimide hydrolysis of a bioconjugate can be monitored by electrospray mass spectrometry since the addition of water to the conjugate results in an increase of 18 Daltons to the observable molecular weight of the conjugate.

Example 11: Cytotoxicity Assay In Vitro

Tumor cells were plated at 2000-5000 cells/well in 180 μL growth medium containing 10% FBS (fetal bovine serum) in 96-well plates and cultured at 37° C. in a humidified incubator with 5% CO₂. After 18 to 24 hours, titrations of antibodies or ADCs in 20 μL were added and cells were incubated for 6 days except for HCC827, NCI-H820, and HEK-293 (5 days) and MIA PaCa2 (4 days). Cell viability was determined using a CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. A non-binding, irrelevant negative control ADC (ADC-4, Structural Formula VIII, where Ab is MSL109-C6v1) was also included in all assays to confirm that cell killing was antigen dependent. All ADCs had an approximately equivalent average DAR.

TABLE 2 Antibody Drug Conjugates Antibody Antibody ADC Heavy Chain Light Chain Structural Conjugation Antibody (SEQ ID NO) (SEQ ID NO) Formula Example ADC-1 AbA  9 10 VIII Ex. 6 ADC-2 AbA  9 10 IX Ex. 7 ADC-3 AbA  9 10 X Ex. 8 ADC-4 MSL109- 11 12 VIII Ex. 9 C6v1

Example 12: Determination of Receptor Density

c-Met cell surface density (antigen binding capacity per cell) was determined by indirect immunofluorescence staining of cell surface antigens on cultured cells using QIFIKIT® (Dako/Agilent). Briefly, cells were harvested from a culture flask as described above for FACS (fluorescence activated cell sorting) analysis, added to a round bottom 96-well plate at 100 μL/well and incubated at 4° C. with 3 μg/mL c-Met antibody m224G11. Wells, treated with an irrelevant mouse monoclonal antibody of the same isotype (mIgG1) at 3 μg/mL, were included as controls. Following a one-hour incubation with primary antibody, cells were centrifuged for 3 minutes at 300×g and washed twice with FACS buffer. For the indirect immunofluorescence staining of the QIFIKIT® beads, 100 μL of resuspended beads from Vial 1 (Set-up Beads) and Vial 2 (Calibration Beads) were added to separate wells, centrifuged for 3 minutes at 300×g, and washed once with FACS buffer. All wells were incubated for one hour at 4° C. with 100 μL of the anti-mouse Alexa Fluor® 488 conjugated antibody (Invitrogen, cat. #A-11029) diluted 1:250 in FACS buffer. Cells were centrifuged for 3 minutes at 300×g, washed twice with FACS buffer, and fixed with 100 μL/well of 1% formaldehyde in PBS (phosphate-buffered saline). Data were acquired on a BD™ LSRII flow cytometer and Geomean values for the 5 bead populations were recorded and used to generate a standard curve based on the lot specific antibody molecules per bead. The standard curve was used to assign ABC (Antibody Binding Capacity or number of receptors) to stained cell samples.

Results

To determine a potential correlation of c-Met expression level and sensitivity to ADC-1, a panel of 9 cell lines were tested in proliferation assays in vitro. FACS analysis demonstrated that these cell lines possess a range of c-Met expression levels as quantified via c-Met antibody binding capacity representing the number of cell surface c-Met molecules (TABLE 3). Sensitivity to ADC-1 in the cell proliferation assay was quantified as maximal killing and IC₅₀ (TABLE 3). ADC-1 inhibited proliferation of cancer cells that over-express c-Met, including the MET amplified cell lines (TABLE 3). As a comparison, unconjugated AbA inhibited proliferation of cells with MET amplification i.e., Hs 746T, SNU-5, and EBC-1 (FIG. 3A-C), but not cell lines without MET amplification, i.e., NCI-H441, NCI-H1573, HCC827, NCI-H820, and Calu-3, and where ADC-1 was also active (FIG. 3D-H). Neither unconjugated AbA nor ADC-1 was active on low-c-Met expressing cells HEK-293 and MIA PaCa2 (TABLE 3, FIG. 31 and FIG. 3J), suggesting that there is a threshold level of c-Met expression required for significant killing by ADC-1.

TABLE 3 Cell Potency In Vitro c-Met Cytotoxicity IC₅₀ (nM) receptors/ ADC-3 ADC-2 ADC-1 Tissue cell^(a) (max^(b)) (max) (max) NSCLC Adenocarcinoma NCI-H441 197,000  0.1 (60%)  0.1 (70%) 0.25 (70%) NCI-H441 170,000  0.2 (85%) 0.25 (90%)  0.3 (90%) NCI-H1573 116,000  1.1 (60%)  1^(c) (40%)  1^(c) (40%) HCC827  94,000  0.5 (80%)  0.9 (70%) 0.85 (70%) NCI-H820  70,000  0.06 (75%) 0.16 (75%) 0.17 (75%) Calu-3  60,000 nd nd   20 (30%) Gastric Hs 746T 320,000 0.050 (40%)  0.1 (50%) 0.17 (50%) SNU-5 290,000  nd^(e) nd 0.19 (90%) Embryonic Kidney HEK-293  27,000 nd nd >60^(d) Pancreatic MIA PaCa-2   5000 >60^(d) >60^(d) >60^(d) ^(a)Approximate number of c-Met molecules on cell surface determined by FACS analysis as antibody binding capacity for m224GI I (the murine parent of AbA) binding at 0.01 mg/mL ^(b)Max is maximum percent decrease relative to the untreated control in the proliferation assay. ^(c)Estimated due to curve-fitting issues ^(d)Not different from control ^(e)nd means not determined

Example 13: c-Met-Targeting TOP1i ADC Inhibits the Growth of Cancer Xenografts In Vivo

The cell lines, Hs 746T and NCI-H441 were obtained from ATCC (American Type Culture Collection). Cells were maintained in monolayer culture for at most 3 passages according to recommendations of the supplier. A suspension of 2×10⁶ cells in culture medium mixed with Matrigel® (1:1, volume:volume) was injected subcutaneously in the right flank of female C.B-17 SCID mice to generate xenografts from the gastric carcinoma cell line, Hs 746T. To generate xenografts from the NSCLC cell line, NCI-H441a suspension of 5×10⁶ cells in culture medium mixed with Matrigel (1:1, volume:volume) was injected subcutaneously in female SCID/bg mice. Treatment started when the size of the flank tumors was approximately 200 mm³.

Vehicle (0 mg/kg) and four dose levels (Doses A-D, from lowest to highest) were administered. Each animal received a single dose.

FIGS. 4A and 4B show ADC-1 inhibited growth of human NSCLC grown as xenografts in immune-compromised mice. Robust growth inhibition is shown after administration of one dose of ADC-1 at Dose C and Dose D, with moderate tumor growth delay after dosing Dose B in both the Hs 746T and NCI-H441 xenografts.

Example 14: c-MET-Targeting TOP1i ADCs Inhibit the Growth of Patient-Derived Cancer Cells In Vivo

The NSCLC patient-derived xenograft (PDX) models, LU450, LU120, LU572, LU123, and LU413, were established internally by implanting patient biopsy tissue fragments subcutaneously in female immunodeficient NOD/SCID mice (Charles River Laboratories). Once tumors were established, PDX tumor cells were expanded by dissociation of tumors to cell suspensions, and 5×10⁴ cells in culture medium were mixed with Matrigel® (1:1, volume:volume) were injected subcutaneously into the mammary fat pad region of female NOD/SCID mice. For efficacy studies, tumor bearing mice were randomized into treatment groups, with each group having equal average tumor volume (130-200 mm³). The c-Met-ADCs were administered as a single dose at six dose levels (doses A-F, from lowest to highest) via intraperitoneal injection. c-Met-targeted efficacy was compared to a similar administration of vehicle. Tumor volumes were measured one to two times weekly, and efficacy was assessed by plotting tumor volume (mm³) versus time to calculate the time to tumor progression (TTP). TTP, expressed in days, is the time after the single dose treatment when tumor regrow to double the size prior to treatment (at randomization). TTP for durable responses (cures) is indicated as the study length with a “>” sign in front of the number.

TABLE 4 In Vivo Activity of ADC-1 and ADC-2 in NSCLC PDX models PDX name ADC-1¹ TTP (days) LU450 0 3 3 15 10 23 LU572 0 9 0.1 26 0.25 >78 1 >91 3 >91 LU211 0 4 3 41 PDX name ADC-2 TTP (days) LU450 0 4 5 34 10 45 LU123 0 8 5 >106 PDX name ADC-1¹ TTP (days) 10 >113 LU120 0 23 5 >94 10 >112 LU413 0 11 5 40 LU413 10 71 ¹0 = vehicle control An ADC having a structure according to structural formula (VIII) demonstrated improved in vivo toxicity.

SEQUENCE LISTING TABLE SEQ ID NO: Description Sequence 1 AbA CDR-H1 GYIFTAYT 2 AbA CDR-H2 IKPNNGLA 3 AbA CDR-H3 ARSEITTEFDY 4 AbA CDR-L1 ESVDSYANSF 5 AbA CDR-L2 RAS 6 AbA CDR-L3 QQSKEDPLT 7 AbA Heavy Chain QVQLVQSGAEVKKPGASVKV Variable Domain SCKASGYIFTAYTMHWVRQA PGQGLEWMGWIKPNNGLANY AQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCARSE ITTEFDYWGQGTLVTVSS 8 AbA Light Chain DIVMTQSPDSLAVSLGERAT Variable Domain INCKSSESVDSYANSFLHWY QQKPGQPPKLLIYRASTRES GVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQSKEDPL TFGGGTKVEIK 9 AbA Heavy Chain QVQLVQSGAEVKKPGASVKV SCKASGYIFTAYTMHWVRQA PGQGLEWMGWIKPNNGLANY AQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCARSE ITTEFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKS CDCHCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSL SLSPG 10 AbA Light Chain DIVMTQSPDSLAVSLGERAT INCKSSESVDSYANSFLHWY QQKPGQPPKLLIYRASTRES GVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQSKEDPL TFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 11 MSL109-C6v1 EEQVLESGGGLVKPGGSLRL Heavy Chain SCAASGFTFSPYSVFWVRQA PGKGLEWVSSINSDSTYKYY ADSVKGRFTISRDNAENSIF LQMNSLRAEDTAVYYCARDR SYYAFSSGSLSDYYYGLDVW GQGTTVIVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 12 MSL109-C6v1 DIVMTQSPLSLSVTPGEPAS Light Chain ISCRSSQSLLHTNGYNYLDW YVQKPGQSPQLLIYLASNRA SGVPDRFSGSGSGTDFTLKI SRVETEDVGVYYCMQALQIP RTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEA 13 m224G11 EVQLQQSGPELVKPGASVKI Heavy SCKTSGYIFTAYTMHWVRQS Chain LGESLDWIGGIKPNNGLANY Variable NQKFKGKATLTVDKSSSTAY Domain MDLRSLTSEDSAVYYCARSE ITTEFDYWGQGTALTVSS 14 m224G11-Light DIVLTQSPASLAVSLGQRAT Chain Variable ISCRASESVDSYANSFMHWY Domain QQKPGQPPKLLIYRASNLES GIPARFSGSGSRTDFTLTIN PVEADDVATYYCQQSKEDPL TFGSGTKLEMK 15 m224G11 GYIFTAYT HC CDR1 16 m224G11 IKPNNGLA HC CDR2 17 m224G11 ARSEITTEFDY HC CDR3 18 m224G11 ESVDSYANSF LC CDR1 19 m224G11 RAS LC CDR2 20 m224G11 QQSKEDPLT LC CDR3 21 DNA encoding atgggatggtcttggatctt AbA Heavy tctgctgtttctgtctggta Chain ctgctggtgtgctgagccag gtccagctggtgcaatccgg cgcagaggtgaagaagccag gcgcttccgtgaaggtgagc tgtaaggcctctggctacat cttcacagcatacaccatgc actgggtgaggcaagctcct gggcagggactggagtggat gggatggattaaacccaaca atgggctggccaactacgcc cagaaattccagggtagggt cactatgacaagggatacca gcatcagcaccgcatatatg gagctgagcaggctgaggtc tgacgacactgctgtctatt attgcgccaggagcgaaatt acaacagaattcgattactg ggggcagggcaccctggtga ccgtgtcctctgccagcacc aagggcccaagcgtgttccc cctggcccccagcagcaaga gcaccagcggcggcacagcc gccctgggctgcctggtgaa ggactacttccccgagcccg tgaccgtgtcctggaacagc ggagccctcacttctggagt tcataccttcccagcagtat tgcagagcagtggcctgtat tcactgtcttccgtcgtaac agttccatcctccagcctcg ggacacagacttacatttgt aacgtgaatcacaagcctag caacaccaaggtcgacaaga gagttgaaccaaagagttgt gattgccactgtcctccctg cccagctcctgagctgcttg gcggtcccagtgtcttcttg tttccccctaaacccaaaga caccctgatgatctcaagga ctcccgaggtgacatgcgtg gtggtggatgtgtctcatga ggacccagaggtgaagttca actggtacgtggacggcgtg gaggtgcacaacgccaagac caagcccagagaggagcagt acaacagcacctacagggtg gtgtccgtgctgaccgtgct gcaccaggactggctgaacg gcaaggagtacaagtgtaag gtgtccaacaaggccctgcc agccccaatcgaaaagacca tcagcaaggccaagggccag ccaagagagccccaggtgta caccctgccacccagcaggg aggagatgaccaagaaccag gtgtccctgacctgtctggt gaagggcttctacccaagcg acatcgccgtggagtgggag agcaacggccagcccgagaa caactacaagaccacccccc cagtgctggacagcgacggc agcttcttcctgtacagcaa gctgaccgtggacaagagca gatggcagcagggcaacgtg ttcagctgctccgtgatgca cgaggccctgcacaaccact acacccagaagagcctgagc ctgtccccaggctga 22 DNA encoding ggctacatcttcacagcata AbA CDR-H1 cacc 23 DNA encoding attaaacccaacaatgggct AbA CDR-H2 ggcc 24 DNA encoding gccaggagcgaaattacaac AbA CDR-H3 agaattcgattac 25 DNA encoding atggaaactgatacactgct AbA Light gctgtgggtcctgctgctgt Chain gggtccctggaagcacaggg gacattgtgatgacccagtc tcccgatagcctggccgtgt ccctgggcgagagggctacc atcaactgtaaaagctccga atctgtggactcttacgcaa acagctttctgcactggtat cagcaaaagccaggccaacc tccaaagctgctgatttaca gggcttctaccagggagagc ggcgtgcccgataggttcag cggatctggcagcggcaccg actttacactgaccatctcc agcctgcaggccgaagatgt ggcagtctattactgccagc agtccaaggaggaccccctg actttcgggggtggtactaa agtggagatcaagcgtacgg tggccgctcccagcgtgttc atcttccccccaagcgacga gcagctgaagagcggcaccg ccagcgtggtgtgtctgctg aacaacttctaccccaggga ggccaaggtgcagtggaagg tggacaacgccctgcagagc ggcaacagccaggagagcgt caccgagcaggacagcaagg actccacctacagcctgagc agcaccctgaccctgagcaa ggccgactacgagaagcaca aggtgtacgcctgtgaggtg acccaccagggcctgtccag ccccgtgaccaagagcttca acaggggcgagtgctga 26 DNA encoding gaatctgtggactcttacgc AbA CDR-L1 aaacagcttt 27 DNA encoding agggcttct AbA CDR-L2 28 DNA encoding cagcagtccaaggaggaccc AbA CDR-L3 cctgact 

1.-4. (canceled)
 5. A method of treating non-small cell lung cancer, the method comprising administering an anti-c-Met antibody drug conjugate comprising the following structure:

wherein n is an integer from 1 to 10, and wherein Ab is an IgG₁ anti-c-Met antibody comprising a heavy chain variable region comprising a heavy chain CDR3 domain comprising the amino acid sequence shown as SEQ ID NO: 3, a heavy chain CDR2 domain comprising the amino acid sequence shown as SEQ ID NO: 2, and a heavy chain CDR1 domain comprising the amino acid sequence shown as SEQ ID NO: 1; and a light chain variable region comprising a light chain CDR3 domain comprising the amino acid sequence shown as SEQ ID NO: 6, a light chain CDR2 domain comprising the amino acid sequence shown as SEQ ID NO: 5, and a light chain CDR1 domain comprising the amino acid sequence shown as SEQ ID NO: 4 to a patient in need thereof. 6.-7. (canceled) 